Rna-mediated gene assembly from dna oligonucleotides

ABSTRACT

The present invention is directed to methods and materials for RNA-mediated gene assembly from oligonucleotide sequences. In some embodiments, the oligonucleotides used for gene assembly are provided in an array format. An RNA polymerase promoter is appended to surface-bound oligonucleotides and a plurality of RNA copies of each oligonucleotide are then produced with an RNA polymerase. These RNA molecules self-assemble into a desired full-length RNA transcript by hybridization and ligation. The resulting RNA transcript may then be converted into double stand DNA useful in a variety of applications including protein expression.

CROSS-REFERENCE TO RELATED APPLICATION

This Divisional patent application claims priority to U.S. Utilitypatent application Ser. No. 13/763,009 filed on Feb. 8, 2013, whichclaims the benefit of U.S. Provisional Patent Application No. 61/597,428filed on Feb. 10, 2012, both of which are incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HG004952 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to methods and materials for RNA-mediatedgene assembly from oligonucleotide sequences on a DNA array. Moreparticularly, an RNA polymerase promoter is appended to surface-boundoligonucleotides, RNA copies are produced using an RNA polymerase, theRNA copies undergo self-assembly and are subsequently ligated to providea full-length target RNA molecule. The RNA molecule is readily copied byRT-PCR to yield the corresponding gene or target double strand DNAfragment.

BACKGROUND OF THE INVENTION

The widespread availability of peptides and oligonucleotides synthesizedby solid-phase chemistries has had a profound impact upon biology andmedicine, with myriad important uses in research, diagnostics, andtherapeutics. A limitation of current technologies is the relativelyshort length of the molecules that can be synthesized, as determined bythe stepwise reaction yield, and thus peptides and oligonucleotides areusually restricted to lengths below ˜50 amino acids or ˜100 nucleotides(nt), respectively. This synthetic limitation has driven interest in thedevelopment of alternative approaches for the production of full-lengthgenes and proteins. The most common strategy has been to splice togethershorter segments into a full-length, functional assembly, for example,the Staudinger ligation reaction permits full-length proteins to beconstructed from a series of peptides (1), and full-length genes can beobtained from multiple short single strands in a series of sequentialligation steps (2) or by Polymerase Cycling Assembly (PCA) (3). However,the assembly-based strategies for gene synthesis reported to date remainlaborious, expensive, and time-consuming, and thus have not yet providedthe level of accessibility needed for widespread utility. As can beappreciated from the above discussion, a need exists for improvedmethods and materials that reduce the labor, expense and time involvedin assembly-based gene synthesis.

SUMMARY OF THE INVENTION

The present invention is based on the inventor's recent discovery of anRNA-mediated assembly method using oligonucleotide sequences on a DNAarray. The inventors' strategy facilitates assembly of full-length RNAtranscripts useful in a variety of life science applications, includinggene synthesis and protein expression.

Accordingly, in a first aspect described herein is a method forRNA-mediated assembly method for providing a target RNA molecule. Such amethod includes steps of: (a) providing an oligonucleotide arraycomprised of: (i) a plurality of first oligonucleotides each having asegment sequence corresponding to a portion of a target RNA and acomplementary RNA Polymerase (RNAP) promoter sequence operably-linked tothe segment sequence's 3′ termini; and (ii) a plurality of secondoligonucleotides each having a splint sequence corresponding to aportion of the target RNA that complements and partially overlaps thesegment sequence of the first oligonucleotides, the secondoligonucleotides including a RNAP promoter sequence operably-linked totheir splint sequence's 3′ termini; (b) hybridizing a thirdoligonucleotide encoding a RNAP promoter sequence to the complementaryRNAP promoter sequence of the first and second pluralities ofoligonucleotides to yield double-stranded RNAP promoters; (c)transcribing with RNA polymerase, in the presence of rNTPs, the segmentsequence of the first plurality of oligonucleotides to yield an RNAsegment and the splint sequence of the second pluralityofoligonucleotides to yield an RNA splint; (d) assembly of the RNAsegments and RNA splints by hybridization to form RNA:RNA hybrids; and(e) sealing nicks in the RNA:RNA hybrid to provide a target RNAmolecule.

In some embodiments, sealing of nicks in the RNA:RNA hybrid is carriedout using any nucleic acid modifying enzyme suitable for ligation ofRNA, such as T4 RNA ligase 2 (or a truncated version). In otherembodiments where 2′-O-Methyl ribonucleotides are used for RNA splintpreparation, sealing of nicks in the RNA:RNA hybrid is carried out usingany nucleic acid modifying enzyme suitable for ligation of RNA with aDNA splint, such as T4 DNA ligase or a truncated version thereof.

In some embodiments, the plurality of first oligonucleotides or theplurality of second oligonucleotides is provided as a surface-boundoligonucleotide array.

In certain embodiments, in step (c), transcription is carried out in thepresence of a mixture of rNTPs and rNMPs.

In other embodiments, the method includes removal of any terminalpyrophosphates from the RNA segments and RNA splints is carried outusing any nucleic acid modifying enzyme suitable for removing suchphosphate moieties, such as 5′ pyrophosphohydrolase or RNApyrophosphatase.

In certain embodiments, the complementary RNAP promoter sequenceoperably-linked to the segment sequence and the splint sequence is acomplementary T7 RNAP promoter sequence or a complementary T3 RNAPpromoter sequence.

In some embodiments of the method, the 5′ end of each segment sequenceand each splint sequence corresponds to a GG dinucleotide in the targetRNA molecule.

The inventive method advantageously allows that steps (d)-(f) in theabove-described method may be, at the discretion of the operator,carried out successively without intervening buffer exchange. Suchoption reduces costs associated with operator labor and time, and costsof reagents and related laboratory materials.

In preferred methods of the invention, the RNA-mediated assembly methodis based on a target RNA molecule that is a full-length RNA transcriptof a gene.

In some embodiments, methods utilize surface-bound oligonucleotideswhich include a spacer, a T7 RNAP promoter sequence, a CC dinucleotideand either the segment sequence or the splint sequence.

In some embodiments, where the method utilizes surface-boundoligonucleotides, the surface-bound oligonucleotides include a 3′ (dT)₁₀spacer, a CTG trinucleotide, a 17mer T7 RNAP promoter sequence, a CCdinucleotide and either the segment sequence or the splint sequence. Inother embodiments, a polyethylene glycol (e.g., PEG-2000) is used as thespacer.

The RNAP promoter sequence contained in the third oligonucleotide is insome cases a T7 RNAP promoter sequence or a T3 RNAP promoter sequence Insome embodiments, the third oligonucleotide includes a T7 RNAP promotersequence and a dinucleotide GG, and in other embodiments comprises atrinucleotide CTG, a 17 mer T7 RNAP promoter sequence and a dinucleotideGG, AG, or a single nucleotide.

In a second and related aspect, the invention provides an RNA-mediatedgene assembly method for providing a target gene. Such a method includessteps of: (a) reverse-transcribing an RNA target molecule provided byany one of the inventive methods described herein; (b) purifying thetarget gene.

In a third and related aspect, the invention provides an RNA-mediatedmethod for providing a target protein. Such a method includes steps of:(a) reverse transcribing an RNA target molecule provided by any one ofthe inventive methods described herein to provide a target gene; (b)expressing a target protein encoded by the target gene; and (c)purifying the target protein.

In another aspect, the invention encompasses an oligonucleotide arrayfor RNA-mediated assembly of a target RNA molecule. Such an arrayincludes: (a) a plurality of first surface-bound oligonucleotides eachhaving a segment sequence corresponding to a portion of a target RNA anda complementary RNAP promoter sequence operably-linked to the segmentsequence's 3′ termini; and (b) a plurality of second surface-boundoligonucleotides each having a splint sequence corresponding to aportion of the target RNA that complements and partially overlaps thesegment sequence of the first surface-bound oligonucleotides, the secondsurface-bound oligonucleotides including a RNAP promoter sequenceoperably-linked to their splint sequence's 3′ termini, wherein the firstand second surface-bound oligonucleotides are linked at their 3′ terminito a surface of the oligonucleotide array.

In preferred embodiments, the target RNA molecule is a full-length RNAtranscript of a gene.

As noted above, a variety of standard and readily obtainable componentsand reagents may be utilized in the combination of inventive steps. Forexample, the oligonucleotide array's surface may be silanized glass or,alternatively, amorphous carbon deposited on a gold film. Accordingly,oligonucleotide arrays useful in the present methods may be provided byany standard fabrication process known in the field including, but notlimited to, in situ photolithographic oligonucleotide array synthesis.

In certain embodiments, the complementary RNAP promoter sequenceoperably-linked to the segment sequence and the splint sequence is acomplementary T7 RNAP promoter sequence or a complementary T3 RNAPpromoter sequence.

In some embodiments, arrays include surface-bound oligonucleotides whichhave a spacer, a 17mer T7 RNAP promoter sequence, a CC dinucleotide andeither the segment sequence or the splint sequence. In some embodiments,arrays include surface-bound oligonucleotides which have a 3′ (dT)₁₀spacer, a CTG trinucleotide, a 17mer T7 RNAP promoter sequence, a CCdinucleotide and either the segment sequence or the splint sequence.Alternatively, the surface-bound oligonucleotides include a PEG-2000instead of (dT)₁₀ as the spacer.

In certain embodiments, the array includes a third oligonucleotide whichhas an RNAP promoter sequence complementary to the RNAP promotersequence of the first and second surface-bound oligonucleotides andwhich hybridizes with those surface-bound oligonucleotides to yielddouble-stranded RNAP promoters. In some embodiments, the thirdoligonucleotide is a T7 RNAP promoter sequence or a T3 RNAP promotersequence, more preferably the third oligonucleotide includes a T7 RNAPpromoter sequence and a dinucleotide GG, AG, or even a single A. In someembodiments comprising arrays, the third oligonucleotide includes atrinucleotide CTG, a 17 mer and a T7 RNAP promoter sequence.

In some embodiments, the first surface-bound oligonucleotides and/or thesecond surface-bound oligonucleotides are bound to the surface of aplurality of beads.

As can be appreciated, the invention encompasses the use ofoligonucleotide arrays as described herein for use in RNA-mediatedassembly of a target RNA molecule. This invention provides the advantageover prior technologies in that embodiments of the invention includefewer manipulation steps and require less operator time than priortechnologies.

In yet another aspect, the present invention is useful for thepreparation of multiple copies of target RNA molecules, including RNApools/libraries. Such oligonucleotide array-based methods to providetarget RNA molecules include steps of: (a) providing an oligonucleotidearray comprised by a plurality of surface-bound oligonucleotides eachhaving a segment sequence corresponding to a portion of a target RNA anda complementary RNAP promoter sequence operably-linked to the segmentsequence's 3′ termini; (b) hybridizing an oligonucleotide encoding aRNAP promoter sequence to the complementary RNAP promoter sequence ofthe surface-bound oligonucleotides to yield double-stranded RNAPpromoters; and (c) transcribing the segment sequence of thesurface-bound oligonucleotide that corresponds to the portion of thetarget RNA sequence with RNA polymerase to yield multiple copies of atarget RNA molecule. In preferred embodiments, a pool of target RNAmolecules differing in nucleotide sequences is provided by the method.

In a related aspect, the invention provides oligonucleotide arraysuseful for carrying out the methods described in the precedingparagraph. Such oligonucleotide arrays include a plurality ofsurface-bound oligonucleotides each having a segment sequencecorresponding to a portion of a target RNA and a complementary RNAPpromoter sequence operably-linked to said segment sequence's 3′ termini.In certain embodiments, the arrays further include an oligonucleotideencoding a RNAP promoter sequence hybridized to the complementary RNAPpromoter sequence of the surface-bound oligonucleotides to yielddouble-stranded RNAP promoters.

In a further aspect, the invention provides an oligonucleotide libraryfor RNA-mediated assembly of a target RNA molecule, where the libraryincludes (a) a plurality of first oligonucleotides each having a segmentsequence corresponding to a portion of a target RNA and a complementaryRNAP promoter sequence operably linked to the segment sequences' 3′termini; and (b) a plurality of second oligonucleotides each having asplint sequence corresponding to a portion of the target RNA thatcomplements and partially overlaps the segment sequence of the pluralityof first oligonucleotides, where the second plurality ofoligonucleotides includes an RNAP promoter sequence operably linked totheir splint sequences' 3′ termini.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the appendedclaims.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the RNA-mediated gene assembly process. A)design of segment (red) and splint (blue) sequences to be employed. B)Segment (dark red) and splint (dark blue) complement sequences assynthesized on the DNA array with the complement of the T7 RNAP promotersequence (green) at their 3′ termini. C) Hybridization of anoligonucleotide encoding the T7 promoter sequence yields the necessarydouble-stranded promoter, and addition of RNA polymerase causestranscription to occur. D) RNA segments and splints have their terminalpyrophosphates removed, assemble by DNA hybridization, and nicks aresealed to yield the desired full-length RNA.

FIG. 2 shows design details of the surface-bound oligonucleotides (theT7 RNAP promoter complement sequence shown at the top of FIG. 2 is SEQID NO:80), along with their complements from solution (the T7 RNAPpromoter sequence in solution shown at the bottom of FIG. 2 is SEQ IDNO:81).

FIG. 3 depicts electropherograms of ZsGreen1 gene and proteinsynthesized from the RNA assemblies. A) Agarose gel electrophoresis ofthe product of RT-PCR amplification of the assembled ZsGreen1 transcriptwith a ZsGreen1 forward primer and a His-tag appended ZsGreen1 reverseprimer, along with a 100 bp DNA ladder marker. The expected size is 714bp. B) Electrophoretic analysis in a “reducing” SDS polyacrylamide gelof the ZsGreen1 protein product obtained in an E. coli cell-freeexpression system from the gene shown in (A). The expected size is 26.9kDa. C) Electrophoretic analysis in a non-reducing SDS polyacrylamidegel of the same protein product and standard marker set shown in (B).ZsGreen1 exists under non-reducing conditions as a tetramer oftheoretical molecular weight 107.6 kDa. It does not migrate true tomolecular weight under these non-reducing gel conditions.

FIG. 4 (A-V) illustrates alignment of Sanger Sequencing Data of ZsGreen1Assemblies ZsGreen1 gene assemblies from DNA arrays fabricated on eitheramorphous carbon surfaces (sequence #1 to #25) or on silanized glasssurfaces (sequence #26 to #51) were Sanger sequenced (FunctionalBiosciences, Inc., WI, USA) and aligned with the ZsGreen1 targetsequence (SEQ ID NO:82; the 714 nucleotide sequence is shown in itsentirety as the top line in FIGS. 4A through 4K, and again as the topline in FIGS. 4L through 4V). It should be noted sequenced nucleotides#1 to #18 correspond to the forward primer sequence (ZsG-F), andsequenced nucleotides #680 to #714 correspond to the reverse primersequence (ZsG-R-w-6His) for ZsGreen1 RT-PCR amplification. Excluding theprimer regions, 16,525 assembled nucleotides from the DNA arrayfabricated on the amorphous carbon surface were analyzed and 25transitions, 3 transversions, 1 deletion, and no insertions wereidentified, which corresponds to an error rate of 0.1755%; whereas17,186 assembled nucleotides from the DNA array on the silanized glasssurface were analyzed and 24 transitions, no transversion, 1 deletion,and 1 insertion were identified, which corresponds to an error rate of0.1513%. This sequence analysis of cloned constructs indicated a yieldof correct constructs of approximately 40%. Analyzing the primersequences (character bordered), which were conventionally columnsynthesized from Sigma Aldrich, 1 transitions, 2 transversions, 3deletions, and 2 insertions were identified in the ZsG-R-w-6His primerregion (35 nt long; 1,785 nucleotides were analyzed; corresponds to anerror rate of 0.448%) whereas no errors were found in the short ZsG-Fprimer region (18 nt long).

FIG. 5 shows images of RT-PCR product bands separated by agarose gelelectrophoresis. The in vitro transcription reaction for segment andsplint RNAs utilized a final concentration of 0.8 mM GMP, 0.1 mM GTP,0.5 mM ATP, 0.5 mM CTP and 0.5 mM UTP along with its reaction buffer andT7 RNA polymerase. The reaction mixture, after buffer exchange, wasdirectly subjected to a ligation reaction with T4 RNA ligase 2.Following, the RNA ligation, the assembled RNA (743 nt) was then RT-PCRamplified with a pair of mWasabi specific primers for a 708 bp longmWasabi gene. The product was analyzed in 2% agarose gel along with aPCR DNA marke ladder (Promega, WI).

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of medicinal chemistry, pharmacology,organic chemistry, analytical chemistry, molecular biology,microbiology, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature.

Gene assembly from DNA arrays was first described in 2004 (4), and hassince been the subject of several other reports (5-10). Its allure liesin the potential to make complete genes as rapidly and inexpensively assingle oligonucleotides are made today, enabled by the ability of DNAarrays to easily provide many thousands of oligonucleotides forassembly. However, gene assembly has remained a costly and laboriousendeavor. Reasons for this include: (a) the oligonucleotides that aresynthesized on DNA arrays must be cleaved from the surface prior to use,and are impure, containing many truncated or chemically modifiedsequences and thus necessitating various labor- and time-intensivepurification or error correction procedures (4,6-10); (b) only minuteamounts of oligonucleotide are made per array feature, necessitatingcomplicated amplification strategies that include adaptor ligation andseveral other steps (4,6-10); (c) virtually all strategies reported todate are based upon Polymerase Cycling Assembly (PCA) (4-11), whichalthough widely used, is complex, laborious, and prone to error (12).

We will now describe an improved method of assembly of full-length RNAtranscripts from DNA array by reference to an exemplary embodiment.Referring to FIG. 1, which illustrates one embodiment of the invention,each element of the DNA array includes a T7 RNA polymerase promotersequence at the 5′ end. Transcription from these surface-bound promotersyields many RNA copies of the oligonucleotide elements encoded in thearray. These amplified RNA molecules self-assemble to yield the desiredfull-length transcript. The transcript, once synthesized, is readilycopied by RT-PCR to yield the corresponding gene.

To provide proof-of-principle, we designed an oligonucleotide array withthe sequences necessary to produce a full-length transcript for thefluorescent protein ZsGreen1. We chose ZsGreen1 for a proof-of-principledemonstration for several reasons: (a) the protein is relatively smallin size, consisting of 231 amino acids; (b) has been shown to foldcorrectly under in vitro translation conditions; and (c) is fluorescentand thus its translation is easily monitored. A full-length RNAtranscript, comprising the 696 nt that encode ZsGreen1 and an additional10 nt corresponding to the Kozak consensus sequence (5′-GGT CGC CAC C-3′(SEQ ID NO:79), added to the 5′ end of the RNA transcript to enhanceeukaryotic in vitro translation efficiency (13)), was assembled fromRNAs produced from photolithographically fabricated oligonucleotidearrays. The 706 nt RNA molecule was divided into 18 segment sequencesand 17 splints, ranging in length from 18 to 58 nt.

FIG. 1 depicts one embodiment of the process of generating RNA sequencesfrom a DNA microarray followed by their assembly and ligation to producethe desired full-length RNA molecule. In this embodiment, the processconsists of six successive steps, as follows: (a) design theoligonucleotide array, (b) fabricate the array; (c) produce many RNAcopies of each array element (“splints” and “segments”, see FIG. 1 andthe text below) using T7 RNA polymerase; (d) remove 5′ terminalpyrophosphates on the splints and segments with RNA 5′pyrophosphohydrolase; (e) allow self-assembly of the splints andsegments into the desired full-length construct by RNA:RNAhybridization; and (f) seal the nicks with T4 RNA ligase 2. This finalRNA product may then be converted into a DNA copy by reversetranscription, whereupon it may be either cloned, or employed directlyto produce more full-length RNAs for in vitro translation or otherpurposes.

Oligonucleotide arrays were designed to encode “segment sequences” whichare the sections of the desired full-length RNA transcript, and “splintsequences” which are complementary RNAs that serve as templates todirect the correct assembly of the RNA segments (FIG. 1A). Twoparameters determined the choice of segment and splint composition inthe exemplary embodiment: first, each was at least about 30 nucleotides(nt) in length, in order to provide at least two 15 nt stretches ofsequence for hybridization during assembly. Second, it was required thatthe 5′ end of each RNA transcript corresponded to a GG dinucleotide,based upon the higher efficiency of transcription exhibited by T7 RNApolymerase when multiple guanine nucleotides are present at the 5′terminus of the transcript being synthesized (see FIG. 1A) (14). GGGtrinucleotide sequences at the 5′ terminus were avoided however, as theyhave been shown to give rise to a ladder of poly G transcripts in whichthe number of G residues can range from 1-3 G, attributed to “slippage”of the enzyme during coupling of GTP (15).

It should be noted that, in general design terms, splint and segmentsequences may be shorter or longer than the particular sequencesdescribed for the exemplary example, and individual segment sequencesmay share less than or more than 15 nucleotides for hybridization withtheir respective splints sequences. It is preferred that the overlapbetween splint and segment sequences should be designed to share anoverlap of about 15 nucleotides on a melting temperature (Tm) normalizedbasis in order to ensure adequate hybridization between respectivesplint and segment sequences.

The design criteria in the exemplary embodiment yielded 18 segmentsequences to encompass the desired 706 nt transcript. Each of the 17splint sequences had a length of 32 nt, corresponding to two 15 ntregions complementary to the segments that it was to join, and anadditional 5′ GG dinucleotide to enhance transcription efficiency. Eachsurface-bound oligonucleotide also included at the 3′ end a 10 base dTspacer sequence (16), and the three base sequence CTG to improve thehybridization stability of the T7 RNA polymerase complement (see below).The overall design of the surface-bound oligonucleotides is illustratedin FIG. 2, and thus consists of five different sequences; a 3′ (dT)10spacer, a CTG trinucleotide, the 17mer T7 promoter sequence, a CCdinucleotide, and finally the desired segment or splint sequence. Inorder to make the necessary double-stranded T7 RNA polymerase promoter,the 22 nt complement (consisting of the three nt CTG complement, the 17nt T7 promoter complement, and the 2 nt GG complement) is included inthe T7 RNA transcription reaction. The addition of RNA polymeraseresults in the synthesis of multiple copies of each RNA segment fromeach oligonucleotide sequence (FIG. 1B).

The DNA arrays used here were in situ synthesized, in a base-by-basemanner, using maskless array synthesizer (MAS) technology (17,18). Thearrays were synthesized on either glass or amorphous carbon substrateswith similar results: silanized glass substrates are the industrystandard for DNA microarrays, whereas we have found that DNA arraysfabricated on amorphous carbon substrates are more stable than theirglass analogs to prolonged incubations at elevated temperatures andrepeated hybridization cycles (19,20).

The fidelity of the oligonucleotide sequences on the microarray isimportant for the correct assembly of a full-length RNA transcript. Thelight-directed synthesis protocols used in this work were thoroughlyoptimized to maximize sequence fidelity and to reduce the number oferrors that occur during array fabrication. Synthesis errors—which canresult in truncates, incorrect sequences, etc.—are not detrimental tohybridization-based assays, but can have adverse consequences in theproduction of useful gene and protein products. The Examples sectionbelow describes the protocols employed in the present work, andhighlights the differences from previously published protocols (18,20).

Milligan et al. have shown that T7 RNA polymerase will produce RNAs fromsingle-stranded synthetic DNA templates having a duplex DNA promoter,producing hundreds to thousands of RNA transcripts per template molecule(14,21). This amplification capability is central to the approachdescribed here, as the increased concentrations of segment and splintstrands drive the hybridization-based assembly process, obviating theneed for further PCR amplification prior to the polymerase cyclingassembly (PCA) employed in all other gene assembly strategies reportedto date (4,6,7,9-11).

The assembly of the RNA segment sequences into the full-length RNAtranscript includes ligation with T4 RNA ligase 2. However, thetranscripts generated by T7 RNA polymerase are triphosphorylated andtherefore must be “trimmed” to their monophosphorylated analogs beforeligation (FIG. 1C). In some embodiments, this is accomplished bytreatment of the transcript pool with RNA 5′ pyrophosphohydrolase (FIG.1C), removing a pyrophosphate group from the 5′ end of each RNA. Inembodiments where monophosphorylated analogs are provided in earliersteps, this trimming step is not necessary. In some embodiments, rNMPsand rNTPs are included at a ratio of about 8:1. In some embodiments, therNMP to be used is GMP, which is included at a ratio of 8:1 relative toGTP, where the other rNTPs are used at their normal concentration forRNA synthesis. Alternatively, mono-phosphorylated RNA transcriptssuitable for ligation may be prepared by manipulations which do notdirectly remove a pyrophosphate group. For example, calf-intestinalalkaline phosphatase (CIP) may be utilized to remove all phosphates fromthe in vitro transcribed RNAs, followed by T4 Polynucleotide Kinase,(PNK) treatment to phosphorylate the RNAs prior to the ligation step.

The assembled RNA segments are then ligated with T4 RNA ligase 2 toproduce the desired full-length transcript. In this embodiment, thepyrophosphate removal and ligation steps utilize a compatible buffer,which permits them to be performed successively in a single tube withoutintervening buffer exchange steps and thereby simplifies the overallassembly process. T4 RNA ligase 2 with ATP is thus added directly intothe RNA 5′ pyrophosphohydrolase-treated reaction, which contains the RNAsegments and splints from the oligonucleotide array. The RNA product wasreverse-transcribed and PCR amplified using forward and reverse primersfor the ZsGreen1 gene. The reverse primer included sequence encoding 6histidines to enable His-tag purification of the protein product (22).

Based on the inventors' efforts described herein, the invention providesin a first aspect an RNA-mediated assembly method for providing a targetRNA molecule. Such a method includes steps of: (a) providing anoligonucleotide array comprised by: (i) a plurality of firstsurface-bound oligonucleotides each having a segment sequencecorresponding to a portion of a target RNA and a complementary RNAPolymerase (RNAP) promoter sequence operably-linked to the segmentsequence's 3′ termini; and (ii) a plurality of second surface-boundoligonucleotides each having a splint sequence corresponding to aportion of the target RNA that complements and partially overlaps thesegment sequence of the first surface-bound oligonucleotides, the secondsurface-bound oligonucleotides including a RNAP promoter sequenceoperably-linked to their splint sequence's 3′ termini, wherein the firstand second surface-bound oligonucleotides are linked at their 3′ terminito a surface of the oligonucleotide array; (b) hybridizing a thirdoligonucleotide encoding a RNAP promoter sequence to the complementaryRNAP promoter sequence of the first and second surface-boundoligonucleotides to yield double-stranded RNAP promoters; (c)transcribing with RNA polymerase the segment sequence of the firstsurface-bound oligonucleotide to yield an RNA segment and the splintsequence of the second surface-bound oligonucleotide to yield an RNAsplint; (d) removing any terminal pyrophosphates from the RNA segmentsand the RNA splints; (e) assembly of the RNA segments and RNA splints byhybridization to form RNA:RNA hybrids; and (f) sealing nicks in theRNA:RNA hybrid to provide a target RNA molecule.

Removal of any terminal pyrophosphates from the RNA segments and RNAsplints is carried out using any nucleic acid modifying enzyme suitablefor removing such phosphate moieties, such as 5′ pyrophosphohydrolase orRNA pyrophosphatase.

Sealing of nicks in the RNA:RNA hybrid is carried out using any nucleicacid modifying enzyme suitable for ligation of RNA, such as T4 RNAligase 2 or a truncated version thereof.

In certain embodiments, the complementary RNAP promoter sequenceoperably-linked to the segment sequence and the splint sequence is acomplementary T7 RNAP promoter sequence or a complementary T3 RNAPpromoter sequence.

In some embodimentsthe 5′ end of each segment sequence and each splintsequence corresponds to a GG dinucleotide in the target RNA molecule.

It is an advantage provided by the invention that steps (d)-(f) in theabove-described method may be, at the discretion of the operator,carried out successively without intervening buffer exchange. Suchoption reduces costs associated with operator labor and time, and costsof reagents and related laboratory materials.

In preferred methods of the invention, the RNA-mediated assembly methodis based on a target RNA molecule that is a full-length RNA transcriptof a gene such that a full-length DNA encoding the gene may ultimatelybe obtained in an expedited manner.

In some embodiments, methods utilize surface-bound oligonucleotideswhich include a spacer, a T7 RNAP promoter sequence, a CC dinucleotideand either the segment sequence or the splint sequence, and particularlypreferred embodiments the surface-bound oligonucleotides further includea trinucleotide CTG and a 17 mer T7 RNAP promoter.

In general, the spacer may vary in length and composition, with suitablelinker/tethering entities constructed from a wide variety of nucleotidesequences, including, e.g., inverted dT (reverse linkage) sequences.Alternatively, spacers useful in the present methods may be constructedfrom non-nucleic acid entities, including but not limited to polymers ofpolyethylene glycol (e.g., PEG18 or PEG2000 spacer arms may be used tosubstitute the spacer in the exemplary embodiment).

The RNAP promoter sequence contained in the third oligonucleotide can bea T7 RNAP promoter sequence or a T3 RNAP promoter sequence. In somecases, the third oligonucleotide includes a T7 RNAP promoter sequenceand a dinucleotide GG, AG, or a single nucleotide (e.g., A). In someembodiments, the third oligonucleotide includes a trinucleotide CTG, a17 mer T7 RNAP promoter sequence and a dinucleotide GG, AG, or a singlenucleotide such as A.

Referring again to the proof-of-principle example, the fidelity of theexemplary assembly process was monitored in four ways. First, the PCRproduct was analyzed by agarose gel electrophoresis. FIG. 3A shows thata single DNA band of the expected size (714 bp) is obtained. Second, thegene was subjected to in vitro translation and the resultant proteinproduct was analyzed by reducing SDS-PAGE electrophoresis. FIG. 3B showsthat only a single band of the expected molecular weight (26,950daltons) is visible by Coomassie Blue staining. Third, the same proteinproduct was analyzed by non-reducing SDS-PAGE electrophoresis anddetected by fluorescence imaging. FIG. 3C shows that only a singlefluorescent protein is observed under these non-reducing electrophoreticconditions. Finally, we cloned the PCR product directly (withoutenzymatic error corrections), and subjected 51 randomly chosen coloniesto Sanger sequencing. 22 of the clone sequences were a perfect match tothe desired target sequence; in total 33,711 bases of DNA sequence wereobtained and 49 transitions, 3 transversions, 2 deletions, and 1insertion were identified (1.63 errors/kb; see Example Section). Thishigh rate of generation of the correct gene sequence (22/51=˜40%) isinvaluable for practical applications of gene synthesis technology.

It can be appreciated that the invention contemplates an RNA-mediatedgene assembly method for providing a target gene. Such a method includessteps of: (a) reverse-transcribing an RNA target molecule provided byany one of the inventive methods described herein; (b) purifying thetarget gene.

In a related aspect, the invention provides an RNA-mediated method forproviding a target protein. Such a method includes steps of: (a) reversetranscribing an RNA target molecule provided by any one of the inventivemethods described herein to provide a target gene; (b) expressing atarget protein encoded by the target gene; and (c) purifying the targetprotein.

In yet another aspect, the invention is directed to the materials usedto carry out the present methods, specifically the uniquely-designedoligonucleotide arrays for RNA-mediated assembly of target RNA moleculesdescribed herein. Such inventive arrays include: (a) a plurality offirst surface-bound oligonucleotides each having a segment sequencecorresponding to a portion of a target RNA and a complementary RNAPpromoter sequence operably-linked to the segment sequence's 3′ termini;and (b) a plurality of second surface-bound oligonucleotides each havinga splint sequence corresponding to a portion of the target RNA thatcomplements and partially overlaps the segment sequence of the firstsurface-bound oligonucleotides, the second surface-boundoligonucleotides including an RNAP promoter sequence operably-linked totheir splint sequence's 3′ termini, wherein the first and secondsurface-bound oligonucleotides are linked at their 3′ termini to asurface of the oligonucleotide array.

In preferred embodiments, the target RNA molecule is a full-length RNAtranscript of a gene.

As noted above, a variety of standard and readily available componentsand reagents may be utilized in the combination of inventive steps. Forexample, the oligonucleotide array's surface may be silanized glass or,alternatively, amorphous carbon deposited on a gold film. Accordingly,oligonucleotide arrays useful in the present methods may be provided byany standard fabrication process known in the field including, but notlimited to, in situ photolithographic oligonucleotide array synthesis.

In certain embodiments, the complementary RNAP promoter sequenceoperably-linked to the segment sequence and the splint sequence is acomplementary T7 RNAP promoter sequence or a complementary T3 RNAPpromoter sequence. In some embodiments the 5′ end of each segmentsequence and each target sequence corresponds to a GG dinucleotide inthe target RNA molecule.

In certain embodiments, arrays include surface-bound oligonucleotideswhich have a 3′ (dT)10 spacer or a PEG 2000 spacer, a CTG trinucleotide,a 17mer T7 RNAP promoter sequence, a CC or other dinucleotide or asingle nucleotide and either the segment sequence or the splintsequence.

In certain embodiments, the array includes a third oligonucleotide whichhas an RNAP promoter sequence complementary to the RNAP promotersequence of the first and second surface-bound oligonucleotides andwhich hybridizes with those surface-bound oligonucleotides to yielddouble-stranded RNAP promoters. The third oligonucleotide, in someembodiments, is a T7 RNAP promoter sequence or a T3 RNAP promotersequence, more preferably the third oligonucleotide includes atrinucleotide CTG, a 17 mer T7 RNAP promoter sequence and a dinucleotideGG, AG, or the single nucleotide A.

In an alternative set of embodiments, segment sequence and splintsequence oligonucleotides are provided in solution as oligonucleotidelibraries, rather than bound to a surface as arrays. The oligo librariescan be similarly used as substrates in the RNA assembly methodsdescribed herein.

In further embodiments, multiple target RNAs may be represented, i.e.,an oligonucleotide array or library can contain segment and splintsequences directed to the assembly of multiple target RNA assemblies. Insuch embodiments, the number of represented RNA sequences may range fromabout 2 to about 100 separate sequences, e.g., 3, 5, 10, 15, 20, 30, 40,50, 60, 65, 70, 75, 80, 90, or another number of represented RNA targetsequences from about 2 to about 100 separate sequences.

In some embodiments, where multiple target RNA sequences arerepresented, the target RNA sequences contain overlapping complementaryends that allow assembly of the separate RNAs into a longer contiguoussequence. Such longer assembled RNA sequences may range from about 1,000bases to about 20,000 bases, e.g., about 1,500, 2,000, 2,500, 3,000,4,000, 5,000, 6,000, 7,000, 10,000, 12,000, 15,000, 17,000, or anothersequence length from about 1000 bases to about 20000 bases. In someembodiments, modular and reiterative use of the methods described hereinallows assembly of much larger contiguous sequences on the order of50,000 bases upwards of about 1 million bases, and ultimately to theassembly of synthetic chromosomes by initial assembly of long RNAtranscripts, reverse transcription and long range PCR or other DNAamplification methods.

In yet other embodiments, where multiple target RNA sequences arerepresented in an array, segment and splint oligonucleotidescomplementary to the same target RNA sequence share at theirsurface-bound 3′ end, in addition to a T7 or a T3 promoter sequence, ashared “tag” sequence of about 10-18 nucleotides, that is uniquelyassociated with oligonucleotides complementary to the same target RNA tobe assembled. To specifically initiate transcription from this subset ofoligonucleotides in this array, an oligo comprising both a 5′-sequencecomplementary to the aforementioned tag sequence and a complementary T7or T3 RNAP promoter sequence to generate an operable T7 or T3 RNAPpromoter sequence is hybridized under stringent conditions so as tohybridize only to the subset of array oligonucleotides containing thecorresponding tag sequence. This system allows the use of a single arraycontaining oligo sets for the assembly of RNAs for multiple genes toassemble individual pre-selected RNA targets by simply adding theappropriately tagged-T7/T3 RNAP oligonucleotide.

Previous work on gene assembly from oligonucleotide arrays has employedthe DNA sequences themselves, rather than assembling RNA intermediatesas employed in this work. The generation of an RNA intermediate hasseveral advantages: (a) ˜100 to 1000 copies of the RNA are produced bytranscription from each DNA strand present on the array (21); thisobviates the need for complex PCR-based oligonucleotide amplification(23) prior to gene assembly (6,7); (b) parallel gene assembly of the RNAsegment and splint sequences, directly from the oligonucleotide array,eliminates a number of laborious steps (e.g., cleavage of theoligonucleotides from the array, amplification of the oligonucleotidepool, and purification of the oligonucleotide pool); (c) the sequencingresults obtained in the present study show that the full-length RNAtranscripts produced have a high sequence fidelity (i.e., a low numberof incorrect sequences), whereas the individual oligonucleotidesproduced during in situ syntheses may include a variety of defects dueto side reactions and incomplete nucleotide coupling reactions (24-27).Sequence errors that are present on the array are presumably copied intothe RNA transcripts; however, these deleterious sequences may beincorporated less often into the full-length RNA transcripts due to theadditional sequence fidelity constraints innate to thehybridization/ligation assembly procedure. (d) The assembled product isan RNA transcript that is readily copied into DNA for cloning or forproduction of more RNA copies by in vitro transcription. TheRNA-mediated assembly process described here is also considerablysimpler and more rapid than previously described multi-step andmulti-day strategies (6,7), involving only four successive enzymaticprocedures that are readily performed in a few hours. Referring to Table1 below, we compare here the RNA-mediated assembly technology with otherrecently published gene assembly technologies. The RNA-mediated strategydrastically reduces the time and labor required for high fidelity genesynthesis from weeks to a days and no specialized equipment is needed(not including the array fabrication). As can be appreciated, thepresent invention provides an avenue to the assembly in a step-wisemanner of large gene clusters, chromosomes, and even eventually genomes.

TABLE 1 Comparison of selected strategies for gene assembly from DNAarrays Kosuri et al. (7) Matzas et al. (6) Wu et al. (this study)Production of oligonucleotide Cleavage of oligos from the T7 RNAamplification from library (Agilent OLS) arrays, and purification. thearray (4 h~12 h)* (proprietary synthesis, cleavage and amplification. 7d)* Assembly -specific PCR Amplification with emulsion PCR Bufferexchange (1 h)* amplification (2 h)* Purification for size verificationSequencing with next generation Pyrophosphate removal (2 h)* sequencerReamplification in 20 mL with Sequences selected and localized T4 RNAligation to produce chemically modified assembly- full-length RNAtranscript specific primers (40 min)* Split into 96 well plates Beadextraction with a micro Reverse transcription PCR actuator from apicotiter plate to for full-length coding gene 96-well plate (3 hr)*Buffer exchange (cleanup) Amplification of DNA individually from beads.Variable biotinylated primers used to remove restriction productscontaining biotin residues by streptavidin matrix. Protease digestionfollowed Gel purification--estimation of by heat inactivation oligoconcentration and mixing the amplicons in equimolar amounts Proteinremoval Removal of primer regions Buffer exchange (cleanup) Ethanolprecipitated Lambda exonuclease digestion Polymerase cycling assemblyBuffer exchange DpnII digestion and USER enzyme (NEB) with guide oligoBuffer exchange (cleanup) Polymerase cycling assembly (4 h)* Errorcorrection (3.5 h)* Column reaction cleanup Restriction digestion (2 h)*Gel purification (2 h)*

We have described here, by reference to an exemplary embodiment, astrategy for the RNA-mediated assembly of full-length RNA transcriptsand, subsequently, a gene from DNA arrays. Proof-of-principle wasdemonstrated in the assembly of a small gene encoding the greenfluorescent protein ZsGreen1, and its in vitro translation to yield afunctional protein. Sequence analysis of cloned constructs indicated ayield of correct constructs of approximately 40%.

Beyond gene assembly, the present invention is also useful for thepreparation of multiple copies of target RNA molecules, including RNApools/libraries. Such oligonucleotide array-based method to providetarget RNA molecules include steps of: (a) providing an oligonucleotidearray comprised by a plurality of surface-bound oligonucleotides eachhaving a segment sequence corresponding to a portion of a target RNA anda complementary RNAP promoter sequence operably-linked to the segmentsequence's 3′ termini; (b) hybridizing an oligonucleotide encoding aRNAP promoter sequence to the complementary RNAP promoter sequence ofthe surface-bound oligonucleotides to yield double-stranded RNAPpromoters; and (c) transcribing the segment sequence of thesurface-bound oligonucleotide that corresponds to the portion of thetarget RNA sequence with RNA polymerase to yield multiple copies of atarget RNA molecule. In preferred embodiments, a pool of target RNAmolecules differing in nucleotide sequences is provided by the method.

In a related aspect, the invention provides oligonucleotide arraysuseful for carrying out the methods described in the precedingparagraph. Such oligonucleotide arrays include a plurality ofsurface-bound oligonucleotides each having a segment sequencecorresponding to a portion of a target RNA and a complementary RNAPpromoter sequence operably-linked to said segment sequence's 3′ termini.In certain embodiments, the arrays further include an oligonucleotideencoding a RNAP promoter sequence hybridized to the complementary RNAPpromoter sequence of the surface-bound oligonucleotides to yielddouble-stranded RNAP promoters. As can be appreciated, thepresently-described arrays find a variety of uses where multiple copiesof RNA molecules are required. Accordingly, this aspect of the inventionmay be utilized, with no more than routine modification, to prepare avariety of RNA-based or related molecules, such as catalytically-activeRNAs (i.e., ribozymes). Alternatively, the inventive methods are usefulfor providing pools/libraries of RNA molecules, such as, e.g., microRNAor siRNA libraries to be screened for desirablebioactivities/functionalities, or, alternatively, for preparingRNA-based probes, including, but not limited to, biotinylated,radio-labeled and fluoro-labeled nucleic acid probes useful in a varietyof detection/imaging applications.

In certain embodiments, the oligonucleotide arrays are provided in theform of a plurality of beads with the above-described oligonucleotidesbound to the surface of such beads covalently or non-covalently.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the disclosed method in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and the following examples andfall within the scope of the appended claims.

III. EXAMPLES Example 1 Generation of Translation-Competent zsGreen RNAby Microarray-Mediated RNA Assembly

Materials and Methods

Design of DNA Arrays for RNA-Mediated Assembly

The full-length ZsGreen1 coding gene plus Kozak sequence (696 nt RNAtranscript encoding ZsGreen1, and 10 additional nucleotidescorresponding to the Kozak sequence for eukaryotic cell-free expressionsystem) was split into 18 segments. Segments were designed to includeterminal GG dinucleotides to enhance in vitro T7 RNA polymerasetranscription. Every segment is longer than 30 nt to provide a minimumof 15 bp hybridization with the splint oligos. Splint RNAs were appendedwith an initial GG dinucleotide for the same reason as for the targetfragments.

37 single stranded DNAs were synthesized on the microarray (see sequenceinformation for details), which includes 18 segment RNA templates, 17splint RNA templates, and two control oligos for quality monitoring. Foreach of the 9 longest segment sequences (>67mer), multiple features weremade rather than just one, in order to increase the amount of RNAproduced (see “sequences on the microarray” below). Each feature issized 1680 μm×1232 μm.

Preparation of Substrates for In Situ Photolithographic OligonucleotideArray Synthesis

Silanized glass. Glass is the standard substrate for DNA arrayfabrication because of its advantages of low intrinsic fluorescence,non-porosity and ease of modification using silane chemistries. Glassmicroscope slides (Plain Micro Slides, VWR, PA, USA) were cleaned with1M sodium hydroxide prior to silanization. The slides were thensilanized for 4 h in 2% (v/v)N-(2-triethoxysilylpropyl)-4-hydroxy-butyramide (Gelest, Inc.,Morrisville, Pa., USA) in stock solution (0.1% acetic acid in 95%ethanol). After being rinsed by stirring in fresh stock solution for 15min, the slides were transferred to a pre-heated (120° C.) oven for 2 h,and cured under vacuum overnight.

Carbon-on-gold. In addition to the use of the above standard glasssubstrates for DNA array fabrication, we also employed substratesoverlaid with amorphous carbon deposited on a gold thin film because oftheir superior thermal stability (18). Tetraethylene glycolmonoallylether was employed for the preparation of hydroxyl terminatedsurfaces for photolithographic oligonucleotide array synthesis since ithas been reported that polyethylene glycol modified surfaces help toreduce nonspecific adsorption of proteins (28). First, standard glassslides coated with 50 Å chromium and 1,000 Å of gold (EMF corp., NY,USA) were extensively rinsed with hexane and ethanol and dried under anitrogen stream. A 7.5 nm layer of amorphous carbon was then DCmagnetron sputtered on the gold surface (Denton Vacuum, NJ, USA). 40 μLof tetraethyleneglycol monoallylether, which was synthesized accordingto a literature procedure (29), was placed directly onto the amorphouscarbon surface, and then covered with a quartz coverslip. The surfaceswere irradiated under nitrogen purge with a low-pressure mercury vaporquartz grid lamp (λ=254 nm, 0.35 mW/cm2) for 16 h. After thephotoreaction, the surfaces were rinsed extensively with ethanol anddeionized water and dried under nitrogen.

In Situ Photolithographic Oligonucleotide Array Synthesis

Light-directed photolithographic synthesis of DNA arrays was performedon either the silanized glass slides or the ethylene glycol modifiedcarbon-on-gold surface with a digital micromirror-based Maskless ArraySynthesis (MAS) system connected to a ABI Expedite™ 8909 Nucleic AcidSynthesis System (Applied Biosystems, CA, USA) as described previously(4,17,30). Oligonucleotide synthesis reagent, 0.1M Activator 42(5-[3,5-Bis(trifluoromethyl)phenyl]-1H-tetrazole) and all NPPOC(3′-nitrophenylpropyloxycarbonyl) protected phosphoramidites[5′-NPPOC-dAdenosine (tac) 3′-β-cyanoethylphosphoramidite (NPPOC-dA),5′-NPPOC-dThymidine 3′-β-cyanoethylphosphoramidite (NPPOC-dT),5′-NPPOC-dCytidine (ib) 3′-β-cyanoethylphosphoramidite (NPPOC-dC),5′-NPPOC-dGuanosine (ipac) 3′-β-cyanoethylphosphoramidite (NPPOC-dG)]were purchased from Sigma Aldrich. Anhydrous wash (acetonitrile),amidite diluent (acetonitrile), capping reagent A (THF/PAc2O), cappingreagent B (Cap Mix B 10% N-Methylimidazole in THF) and deblocking mix(3% dichloroacetic acid in dichloromethane) were purchased from GlenResearch (VA, USA). Oxidizing reagent (0.02 M iodine inTHF/pyridine/H2O, 89.6/0.4/10) was purchased from EMD Chemicals (NJ,USA). Exposure solvent is 1% imidazole in DMSO. Anhydrous reagents werekept over molecular sieves (AldraSORB™ water trapping packets, SigmaAldrich). The oligonucleotide synthesis protocol was modified andoptimized based on previously published protocols. (18,20) Briefly,every synthesis cycle contains two capping steps to achieve high yieldof full-length templates and one oxidation to stabilize thephosphodiester bonds. The step-sequence was coupling (80 sec), capping(20 sec), oxidizing (15 sec), capping (flow though), and UVdeprotection. The light dose to remove the photolabile NPPOC(3′-nitrophenylpropyloxycarbonyl)-protecting groups from NPPOCphosphoramidites (Sigma Aldrich, MO, USA) was determined prior to DNAarray fabrication. A series of incremental doses of 365 nm light(Joule/cm2) was used for a 30 nt quality control (QC) oligonucleotidesynthesis. The optimal dose was chosen to yield the highest level offluorescence from hybridization of a fluorescently tagged QC complement.It is noted that the complete removal of NPPOC protecting group isimportant to eliminate possible deletions during synthesis. Arraysynthesis proceeded as follows: (a) after coupling of the previousNPPOC-protected base to the growing DNA strand, the synthesis flow cell(volume˜100 μl) was flushed with 500 μl of exposure solvent; (b) adigital image (mask) representing the locations for the next baseaddition illuminated the surface with either 4.2 Joule/cm2 of 365 nmlight on silanized glass or 3.5 Joule/cm2 of 365 nm on carbon surfaceusing a 350 watt mercury arc lamp (Newport, Conn., USA). Exposuresolvent was constantly flowed through the flow cell at a rate of 180˜220μl/(Joule/cm2) during illumination, sufficiently maintaining the basicconditions needed to drive the photocatalyzed elimination reaction. (31)(c) Following irradiation, the array was washed with acetonitrile (˜400μl) to remove residual exposure solvent, dry wash (˜300 μl) to removetrace water, and activator solution (˜100 μl). (d) Coupling of the nextbase was achieved by filling the flow cell with a 1:1 solution of thedesired phosphoramidite and Activator 42. All 5′-NPPOC-protectedamidites underwent a single 80 s coupling step. (e) After amiditecoupling, the array was capped with a 1:1 v/v mixture of cappingreagents A and B (A:B solution) for 20 sec (˜320 μl). (d) After washingwith acetonitrile (˜100 μl) the array was oxidized with oxidizersolution for 15 sec (THF, pyridine, iodine, and water, ˜480 μl). (e) Asecond capping step was performed by flushing the cell with cappingreagent A:B solution. (f) After synthesis is complete, the nucleosidebases are deprotected in 1:1 ethylenediamine:absolute ethanol solutionat room temperature for 2 hr. The primary significant differences frompreviously published protocols (18,20) are: (i) a higher photo dose wasused to remove the NPPOC-protecting groups on the carbon-on-goldsurface; (ii) a longer coupling time (80 sec) and different activator(Activator 42) were used; (iii) capping was conducted directly aftereach amidite coupling followed by oxidation and another capping step;(iv) An oxidizing step was included in every cycle.

On-Chip RNA Transcription with T7 RNA Polymerase

A gasket, Gene Frame—15×16 mm internal (Abgene, Epsom, UK), was attachedand surrounded the DNA features. A 100 μl total in vitro RNAtranscription reaction contains a final concentration of 2.25 U/μl T7RNA polymerase-Plus™ (Ambion, TX, USA), 0.8 mM each NTP, 1 μM T7RNAPpromoter complement, Ix RNAsecure™ reagent (Ambion, TX, USA), 20 mMNaCl, 40 mM Tris pH 7.8, 6 mM MgCl2 2 mM spermidine, and 10 mM DTT. Thereaction mixture, except T7 RNA polymerase, was applied to the chip andincubated at 60° C. for 10 min, then slowly cooled down to roomtemperature. T7 RNA polymerase was then added to the surface. Thetranscription reaction was conducted at room temperature for 4˜12 hr ina humid chamber. The total reaction was collected and desalted threetimes with deionized water using a cellulose-based 3,000 molecularweight cut-off Amicon Ultra-0.5 mL centrifugal filter (Millipore, Mass.,USA) prior to pyrophosphate removal.

Pyrophosphate Removal from Triphosphorylated RNA Transcripts

RNA transcripts initiated with triphosphorylated GG dinucleotides weretreated with RNA 5′ Pyrophosphohydrolase (RppH) (NEB, MA, USA) inamended T4 RNA ligase 2 reaction buffer (without ATP) instead of1×NEBuffer 2 (NEB) to reduce the possibility of losses due to extrasteps, and to simplify the overall assembly process. 5 units of RppHwere used to remove pyrophosphate group in a half volume of bufferexchanged RNA transcription reaction in a final concentration of 50 mMTris-HCl pH7.5, 2 mM MgCl2, and 1 mM DTT. The reaction was incubated at37° C. for 2 hr in a total volume of 25 μl.

Full-Length RNA Ligation with T4 RNA Ligase 2

10 units of T4 RNA ligase 2 and a final concentration of 800 μM ATP wereadded to the RppH treated reaction above (a half of the total on chiptranscribed RNAs.) The ligation reaction involved an initial ligationstep at 37° C. for 10 min, followed by 3 cycles of thermal-cycledligation at 65° C. for 30 sec and 37° C. for 5 min, and finished with afinal ligation step at 37° C. for 10 min.

10 units of T4 RNA ligase 2 and a final concentration of 800 μM ATP wereadded to the RppH treated reaction above (a half of the total on chiptranscribed RNAs.) The ligation reaction involved an initial ligationstep at 37° C. for 10 min, followed by 3 cycles of thermal-cycledligation at 65° C. for 30 sec and 37° C. for 5 min, and finished with afinal ligation step at 37° C. for 10 min. The reaction temperature forligation could also be at a fixed temperature of 37° C. for 50 min.

In some cases, the guanosine-initiating T7 class III promoter phi 6.5 isreplaced with the adenosine-initiating T7 class II promoter phi2.5 todecrease 5′ heterogeneity of RNA transcripts. In addition, thereplacement of T7 RNA promoter provides certain degree of flexibilityfor experiment design, i.e., segment and splint RNAs will be free ofrestriction to initiate with guanosine. Also, the penultimatedeoxyribonucleotide of DNA template could be replaced with a C2′ methoxyRNA ribonucleotide to reduce 3′ heterogeneity of RNA transcripts in atranscription reactio, which is deleterious to specific RNA ligationreactions in this method.

In some cases, pyrophosphate removal from triphosphorylated RNAtranscripts is not necessary. The 5′ monophosphorylated RNA transcriptscan be prepared by including excess guanosine monophosphate (GMP) in thetranscription reaction. GMP is only incorporated at 5′-end of thetranscript. Ideally, a high proportion of 5′ monophosphorylated RNAtranscript will result from skewing the ratio of GMP to GTP, e.g., at aratio of GMP to GTP of 8:1. The product is then subjected to a ligationreaction with T4 RNA ligase 2, as shown in FIG. 5. An example wasprovided (Figure ?).

Reverse Transcription PCR for Assembled RNA Transcripts

Assembled ZsGreen1 RNA transcripts for cloning and prokaryotic cell-freeprotein expression were amplified by reverse transcription PCR (RT-PCR)using a OneStep RT-PCR Kit (QIAGEN, CA, USA). ZsGreen1 specific primers,ZsG-F and ZsG-R-w-6His were used (see Sequence Information for details).Cycling consisted of 30 min at 50° C., 15 min at 95° C.; then 40 cyclesof 30 sec at 95° C., 30 sec at 61° C., and 1 min at 72° C.; and finalelongation 10 min at 72° C.

Assembled ZsGreen1 RNA transcripts for eukaryotic cell-free proteinexpression were amplified by RT-PCR using a GeneAmp Gold RNA PCR ReagentKit (Applied Biosystems, CA, USA). ZsGreen1 specific primers: ZsG-F andZsG-R were used. Cycling consisted of 12 min at 42° C., 10 min at 95°C.; then 45 cycles of 20 sec at 94° C., 20 sec at 58° C., and 30 min at72° C.; and final elongation 7 min at 72° C. The ZsGreen1 DNA was gelpurified. Next, T7-ZsG-F and ZsG-R primers were used to append a T7promoter to ZsGreen1 coding gene. Phusion Hot Start High-Fidelity DNAPolymerase (NEB) was used. Cycling consisted of 30 sec at 98° C.; then35 cycles of 10 sec at 98° C., 20 sec at 62° C., and 30 sec at 72° C.;and final elongation 10 min at 72° C.

The RT-PCR products were analyzed by electrophoresis in a 1.5% agarosegel along with a 100 bp DNA ladder (NEB).

Cell-Free Protein Expression, Purification and Detection

Prokaryotic Cell-Free Protein Expression.

The assembled ZsGreen1 gene without Kozak sequence was ligated topEXP5-CT/TOPO vector (Invitrogen, OR, USA) followed by transformationinto ONE Shot TOP10 Competent E. coli cells (Invitrogen). The plasmidswith inserts were purified with a QIAprep Spin Miniprep Kit (QIAGEN).One microgram of plasmid DNA was used in a standard 100 μl reaction ofExpressway Mini Cell-Free E. coli Expression System (Invitrogen). Theprotein expression reaction was performed for 4 hr at 30° C. ZsGreen1protein was either directly analyzed in protein gels or purified withNi-NTA Magnetic Agarose Beads (QIAGEN) prior to the analysis.

Eukaryotic Cell-Free Protein Expression.

ZsGreen1 RNA transcripts with Kozak sequence were produced fromassembled T7 promoter appended ZsGreen1 gene by using a MEGAscript T7kit (Ambion). The transcription reactions were buffer exchanged withwater using a cellulose-based 30,000 molecular weight cut-off AmiconUltra-0.5 mL centrifugal filter. Approximately 3.7 micrograms of RNAtranscripts were used in a 20 μl Retic Lysate IVT (Ambion) cell-freeexpression reaction.

Protein Analysis.

The protein products obtained from the in vitro expression system wereanalyzed in either reducing (a final concentration of 2.5%beta-mercaptoethanol was added to denature the samples at 95° C. for 5min), or non-reducing gradient SDS-PAGE gels (4-20%, Bio-Rad, Richmond,Calif., USA). The prestained broad range protein standard marker (7-175kDa) run along with the protein samples in the SDS-PAGE gel waspurchased from NEB. The reducing SDS-PAGE gels were visualized byCoomassie Blue staining. The fluorescent proteins in the non-reducingSDS-PAGE gels were visualized under a 488 nm laser with a 530 nm filterusing a Bio-Rad Molecular Imnager FX Pro.

Sequence Information

Target Sequence (ZsGreen1, Adapted from Clontech's pZsGreen1-C1 Vector)

Note: The underscored region is the Kozak sequence. The initial GG isincluded in the T7 RNAP transcript for better transcription efficiency.

SEQ ID NO. 1 GGTCGCCACCATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTCGACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGTCCAAGTTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCCCTCCTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATCTTGAAGGGCGACGTGAGCATGTACCTGCTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGAACCAGAAGTGGCACCTGACCGAGCACGCCATCGCCT CCGGCTCCGCCTTGCCCTGA

Target Segment-RNAs for ZsGreen1 Assembly (5′ to 3′)

SEQ ID NO. 2  1 GGUCGCCACCAUGGCCCAGUCCAAGCACGGCC UGACCAA SEQ ID NO. 3  2GGAGAUGACCAUGAAGUACCGCAUGGAGGGCU GCGU SEQ ID NO. 4  3GGACGGCCACAAGUUCGUGAUCACCGGCGA SEQ ID NO. 5  4GGGCAUCGGCUACCCCUUCAAGGGCAAGCA SEQ ID NO. 6  5GGCCAUCAACCUGUGCGUGGUGGAGGGCGGCC CCUUGCCCUUCGCCGA SEQ ID NO. 7  6GGACAUCUUGUCCGCCGCCUUCAUGUACGGCA ACCGCGUGUUCACCGAGUACCCCCA SEQ ID NO. 8 7 GGACAUCGUCGACUACUUCAAGAACUCCUGCC CCGCC SEQ ID NO. 9  8GGCUACACCUGGGACCGCUCCUUCCUGUUCGA SEQ ID NO. 10  9GGACGGCGCCGUGUGCAUCUGCAACGCCGACA UCACCGUGAGCGU SEQ ID NO. 11 10GGAGGAGAACUGCAUGUACCACGAGUCCAAGU UCUAC SEQ ID NO. 12 11GGCGUGAACUUCCCCGCCGACGGCCCCGUGAU GAAGAAGAUGACCGACAACU SEQ ID NO. 13 12GGGAGCCCUCCUGCGAGAAGAUCAUCCCCGUG CCCAAGCA SEQ ID NO. 14 13GGGCAUCUUGAAGGGCGACGUGAGCAUGUACC UGCUGCUGAA SEQ ID NO. 15 14GGACGGUGGCCGCUUGCGCUGCCAGUUCGACA CCGUGUACAA SEQ ID NO. 16 15GGCCAAGUCCGUGCCCCGCAAGAUGCCCGACU SEQ ID NO. 17 16GGCACUUCAUCCAGCACAAGCUGACCCGCGA SEQ ID NO. 18 17GGACCGCAGCGACGCCAAGAACCAGAAGUGGC ACCUGACCGAGCACGCCAUCGCCUCCSEQ ID NO. 19 18 GGCUCCGCCUUGCCCUGA

Target Splint-RNAs for ZsGreen1 Assembly (5′ to 3′)

SEQ ID NO. 20  1 GGGCACGGCCUGACCAAGGAGAUGACCAUGAA SEQ ID NO. 21  2GGCAUGGAGGGCUGCGUGGACGGCCACAAGUU SEQ ID NO. 22  3GGCGUGAUCACCGGCGAGGGCAUCGGCUACCC SEQ ID NO. 23  4GGCUUCAAGGGCAAGCAGGCCAUCAACCUGUG SEQ ID NO. 24  5GGCUUGCCCUUCGCCGAGGACAUCUUGUCCGC SEQ ID NO. 25  6GGCACCGAGUACCCCCAGGACAUCGUCGACUA SEQ ID NO. 26  7GGAACUCCUGCCCCGCCGGCUACACCUGGGAC SEQ ID NO. 27  8GGCUCCUCCUGUUCGAGGACGGCGCCGUGUG SEQ ID NO. 28  9GGCAUCACCGUGAGCGUGGAGGAGAACUGCAU SEQ ID NO. 29 10GGGAGUCCAAGUUCUACGGCGUGAACUUCCCC SEQ ID NO. 30 11GGAGAUGACCGACAACUGGGAGCCCUCCUGCG SEQ ID NO. 31 12GGCCCCGUGCCCAAGCAGGGCAUCUUGAAGGG SEQ ID NO. 32 13GGGUACCUGCUGCUGAAGGACGGUGGCCGCUU SEQ ID NO. 33 14GGCGACACCGUGUACAAGGCCAAGUCCGUGCC SEQ ID NO. 34 15GGGCAAGAUGCCCGACUGGCACUUCAUCCAGC SEQ ID NO. 35 16GGCAAGCUGACCCGCGAGGACCGCAGCGACGC SEQ ID NO. 36 17GGCACGCCAUCGCCUCCGGCUCCGCCUUGCCC

RT-PCR Primer Sequences for Cloning and Sequencing:

SEQ ID NO. 37 ZsG-F: ATGGCCCAGTCCAAGCAC (Sigma Aldrich, MO, USA)SEQ ID NO. 38 ZsG-R-w-6His: CTAGTGGTGATGGTGATGATGGGGCAAGGCGGAGC(Sigma Aldrich, MO, USA)

RT-PCR Primer Sequences for Amplification of T7 Promoter AppendedZsGreen1 Gene:

SEQ ID NO. 39 T7P-ZsG-F:CAGTAATACGACTCACTATAGGTCGCCACCATGGCCCAGTCCAAGCACG(Sigma Aldrich, MO, USA) SEQ ID NO. 40 ZsG-R: TCAGGGCAAGGCGGAGC(Sigma Aldrich, MO, USA)

Complementary Sequence to 77 RNA Polymerase Promoter for In Vitro RNATranscription:

SEQ ID NO. 41 FAM(6-carboxyfluorescein)-CAGTAATACGACTCACTATAGG((Integrated DNA Technologies, IA, USA)

Sequences on the Microarray (5′→3′):

Note: 3′ tethered on the array surface. There are 18 segmented oligos,17 splint oligos, and 2 quality control oligos. Multiple duplicatefeatures were made as marked. Each feature is sized 1680 μm×1232 μm.

SEQ Copy ID NO. # Sequences 42  1 1 CCACTGTTGCAAAGTTATACTCTTGCAGGTCATCGGCCTTTTTTTTTT(QC) 43  2 1 ACTCTTGCAGGTCACGGCCCACTGTTGCAAAGTTATCCTTTTTTTTTT(QC) 44  3 3 TTGGTCAGGCCGTGCTTGGACTGGGCCATGGTGGCGACCTATAGTGAGTCGTATTACTGTTTTTTTTTT (seg) 45  4 1ACGCAGCCCTCCATGCGGTCTTCATGGTCATCTCC TATAGTGAGTCGTATTACTGTTTTTTTTTT(seg)46  5 1 TCGCCGGTGATCACGAACTTGTGGCCGTCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(seg) 47  6 1TGCTTGCCCTTGAAGGGGTAGCCGATGCCCTATAG TGAGTCGTATTACTGTTTTTTTTTT(seg) 48  75 TCGGCGAAGGGCAAGGGGCCGCCCTCCACCACGCACAGGTTGATGGCCTATAGTGAGTCGTATTACTGTT TTTTTTTT(seg) 49  8 5TGGGGGTACTCGGTGAACACGCGGTTGCCGTACAT GAAGGCGGCGGACAAGATGTCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(seg) 50  9 1 GGCGGGGCAGGAGTTCTTGAAGTAGTCGACGATTTCCTATAGTGAGTCGTATTACTGTTTTTTTTTT (seg) 51 10 1TCGAACAGGAAGGAGCGGTCCCAGGTGTAGCCTAT AGTGAGTCGTATTACTGTTTTTTTTTT(seg) 5211 3 ACGCTCACGGTGATGTCGGCGTTGCAGATGCACACGGCGCCGTCCTATAGTGAGTCGTATTACTGTTTTT TTTTT(seg) 53 12 2GTAGAACTTGGACTCGTGGTACATGCAGTTCTCCT CCTATAGTGAGTCGTATTACTGTTTTTTTTTT(seg) 54 13 5 AGTTGTCGGTCATCTTCTTCATCACGGGGCCGTCGGCGGGGAAGTTCACGCCTATAGTGAGTCGTATTAC TGTTTTTTTTTT(seg) 55 14 3TGCTTGGGCACGGGGATGATCTTCTCGCAGGAGGG CTCCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(seg) 56 15 3 TTCAGCAGCAGGTACATGCTCACGTCGCCCTTCAAGATGCCCTATAGTGAGTCGTATTACTGTTTTTTTT TT(seg) 57 16 3TTGTACACGGTGTCGAACTGGCAGCGCAAGCGCCA CCGTCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(seg) 58 17 1 AGTCGGGCATCTTGCGGGGCACGGACTTGGCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(seg) 59 18 1TCGCGGGTCAGCTTGTGCTGGATGAAGTGCCTATA GTGAGTCGTATTACTGTTTTTTTTTT(seg) 6019 5 GGAGGCGATGGCGTGCTCGGTCAGGTGCCACTTCTTTCTTGGCGTCGCTGCGGTCCTATAGTGAGTCGTA TTACTGTTTTTTTTTT(seg) 61 20 1TCAGGGCAAGGCGGAGCCTATAGTGAGTCGTATTA CTGTTTTTTTTTT(seg) 62 21 1GCACGGCCTGACCAAGGAGATGACCATGAACCTAT AGTGAGTCGTATTACTGTTTTTTTTTT(splint)63 22 1 CATGGAGGGCTGCGTGGACGGCCACAAGTTCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 64 23 1CGTGATCACCGGCGAGGGCATCGGCTACCCTATAG TGAGTCGTATTACTGTTTTTTTTTT(splint) 6524 1 CTTCAAGGGCAAGCAGGCCATCAACCTGTGCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 66 25 1CTTGCCCTTCGCCGAGGACATCTTGTCCGCCTATA GTGAGTCGTATTACTGTTTTTTTTTT(splint)67 96 1 CACCGAGTACCCCCAGGACATCGTCGACTACCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 68 27 1AACTCCTGCCCCGCCGGCTACACCTGGGACCTATA GTGAGTCGTATTACTGTTTTTTTTTT(splint)69 28 1 CTCCTTCCTGTTCGAGGACGGCGCCGTGTGCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 70 29 1CATCACCGTGAGCGTGGAGGAGAACTGCATCCTAT AGTGAGTCGTATTACTGTTTTTTTTTT(splint)71 30 1 GAGTCCAAGTTCTACGGCGTGAACTTCCCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 72 31 1AGATGACCGACAACTGGGAGCCCTCCTGCGCCTAT AGTGAGTCGTATTACTGTTTTTTTTTT(splint)73 32 1 CCCCGTGCCCAAGCAGGGCATCTTGAAGGGCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 74 33 1GTACCTGCTGCTGAAGGACGGTGGCCGCTTCCTAT AGTGAGTCGTATTACTGTTTTTTTTTT(splint)75 34 1 CGACACCGTGTACAAGGCCAAGTCCGTGCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 76 35 1AAGATGCCCGACTGGCACTTCATCCAGCCTATAGT GAGTCGTATTACTGTTTTTTTTTT(splint) 7736 1 CAAGCTGACCCGCGAGGACCGCAGCGACGCCTATAGTGAGTCGTATTACTGTTTTTTTTTT(splint) 76 37 1CACGCCATCGCCTCCGGCTCCGCCTTGCCCTATAG TGAGTCGTATTACTGTTTTTTTTTT(splint)

Alignment of Sanger Sequencing Data of ZsGreen1 Assemblies

ZsGreen1 gene assemblies from DNA arrays fabricated on either amorphouscarbon surfaces (sequence #1 to #25) or on silanized glass surfaces(sequence #26 to #51) were Sanger sequenced (Functional Biosciences,Inc., WI, USA) and aligned with the ZsGreen1 target sequence (see FIG.4A-L). It should be noted sequenced nucleotides #1 to #18 correspond tothe forward primer sequence (ZsG-F), and sequenced nucleotides #680 to#714 correspond to the reverse primer sequence (ZsG-R-w-6His) forZsGreen1 RT-PCR amplification. Excluding the primer regions, 16,525assembled nucleotides from the DNA array fabricated on the amorphouscarbon surface were analyzed and 25 transitions, 3 transversions, 1deletion, and no insertions were identified, which corresponds to anerror rate of 0.1755%; whereas 17,186 assembled nucleotides from the DNAarray on the silanized glass surface were analyzed and 24 transitions,no transversion, 1 deletion, and 1 insertion were identified, whichcorresponds to an error rate of 0.1513%. This sequence analysis ofcloned constructs indicated a yield of correct constructs ofapproximately 40%.

Analyzing the primer sequences (character bordered), which wereconventionally column synthesized from Sigma Aldrich, 1 transitions, 2transversions, 3 deletions, and 2 insertions were identified in theZsG-R-w-6His primer region (35 nt long; 1,785 nucleotides were analyzed;corresponds to an error rate of 0.448%) whereas no errors were found inthe short ZsG-F primer region (18 nt long).

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

REFERENCES

-   1. Nilsson, B. L., Soellner, M. B. and Raines, R. T. (2005) Chemical    synthesis of proteins. Annu. Rev. Biophys. Biomolec. Struct., 34,    91-118.-   2. Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heyneker, H. L.,    Bolivar, F. and Boyer, H. W. (1977) Expression in Escherichia coli    of a chemically synthesized gene for the hormone somatostatin.    Science, 198, 1056-1063.-   3. Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M. and    Heyneker, H. L. (1995) Single-step assembly of a gene and entire    plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164,    49-53.-   4. Richmond, K. E., Li, M. H., Rodesch, M. J., Patel, M., Lowe, A.    M., Kim, C., Chu, L. L., Venkataramaian, N., Flickinger, S. F.,    Kaysen, J. et al. (2004) Amplification and assembly of chip-eluted    DNA (AACED): a method for high-throughput gene synthesis. Nucleic    Acids Res, 32, 5011-5018.-   5. Quan, J. Y., Saaem, I., Tang, N., Ma, S. M., Negre, N., Gong, H.,    White, K. P. and Tian, J. D. (2011) Parallel on-chip gene synthesis    and application to optimization of protein expression. Nat.    Biotechnol., 29, 449-U239.-   6. Matzas, M., Stahler, P. F., Kefer, N., Siebelt, N., Boisguerin,    V., Leonard, J. T., Keller, A., Stahler, C. F., Haberle, P.,    Gharizadeh, B. et al. (2010) High-fidelity gene synthesis by    retrieval of sequence-verified DNA identified using high-throughput    pyrosequencing. Nat. Biotechnol., 28, 1291-U1101.-   7. Kosuri, S., Eroshenko, N., LeProust, E. M., Super, M., Way, J.,    Li, J. B. and Church, G. M. (2010) Scalable gene synthesis by    selective amplification of DNA pools from high-fidelity microchips.    Nat. Biotechnol., 28, 1295-U1108.-   8. Borovkov, A. Y., Loskutov, A. V., Robida, M. D., Day, K. M.,    Cano, J. A., Le Olson, T., Patel, H., Brown, K., Hunter, P. D. and    Sykes, K. F. (2010) High-quality gene assembly directly from    unpurified mixtures of microarray-synthesized oligonucleotides.    Nucleic Acids Res, 38, e180.-   9. Kim, C., Kaysen, J., Richmond, K., Rodesch, M., Binkowski, B.,    Chu, L., Li, M., Heinrich, K., Blair, S., Belshaw, P. et al. (2006)    Progress in gene assembly from a MAS-driven DNA microarray.    Microelectron. Eng., 83, 1613-1616.-   10. Tian, J. D., Gong, H., Sheng, N. J., Zhou, X. C., Gulari, E.,    Gao, X. L. and Church, G. (2004) Accurate multiplex gene synthesis    from programmable DNA microchips. Nature, 432, 1050-1054.-   11. Xiong, A. S., Yao, Q. H., Peng, RH., Li, X., Fan, H. Q.,    Cheng, Z. M. and Li, Y. (2004) A simple, rapid, high-fidelity and    cost-effective PCR-based two-step DNA synthesis method for long gene    sequences. Nucleic Acids Res, 32, e98.-   12. Xiong, A. S., Peng, RH., Zhuang, J., Liu, J. G., Gao, F.,    Chen, J. M., Cheng, Z. M. and Yao, Q. H. (2008)    Non-polymerase-cycling-assembly-based chemical gene synthesis:    strategies, methods, and progress. Biotechnol. Adv., 26, 121-134.-   13. Kozak, M. (1987) An analysis of 5′-noncoding sequences from 699    vertebrate messenger RNAs. Nucleic Acids Res, 15, 8125-8148.-   14. Milligan, J. F., Groebe, D. R., Witherell, G. W. and    Uhlenbeck, O. C. (1987) Oligoribonucleotide Synthesis Using T7    Rna-Polymerase and Synthetic DNA Templates. Nucleic Acids Res, 15,    8783-8798.-   15. Martin, C. T., Muller, D. K. and Coleman, J. E. (1988)    Processivity in early stages of transcription by T7 RNA polymerase.    Biochemistry, 27, 3966-3974.-   16. Guo, Z., Guilfoyle, R. A., Thiel, A. J., Wang, R. F. and    Smith, L. M. (1994) Direct fluorescence analysis of genetic    polymorphisms by hybridization with oligonucleotide arrays on glass    supports. Nucleic Acids Res, 22, 5456-5465.-   17. Singh-Gasson, S., Green, R. D., Yue, Y. J., Nelson, C.,    Blattner, F., Sussman, M. R. and Cerrina, F. (1999) Maskless    fabrication of light-directed oligonucleotide microarrays using a    digital micromirror array. Nat. Biotechnol., 17, 974-978.-   18. Phillips, M. F., Lockett, M. R., Rodesch, M. J., Shortreed, M.    R., Cerrina, F. and Smith, L. M. (2008) In situ oligonucleotide    synthesis on carbon materials: stable substrates for microarray    fabrication. Nucleic Acids Res, 36, e7.-   19. Lockett, M. R. and Smith, L. M. (2009) Fabrication and    characterization of DNA arrays prepared on carbon-on-metal    substrates. Anal Chem, 81, 6429-6437.-   20. Lockett, M. R., Weibel, S. C., Phillips, M. F., Shortreed, M.    R., Sun, B., Corn, R. M., Hamers, R. J., Cerrina, F. and    Smith, L. M. (2008) Carbon-on-metal films for surface plasmon    resonance detection of DNA arrays. Journal of the American Chemical    Society, 130, 8611-8613.-   21. Milligan, J. F. and Uhlenbeck, O. C. (1989) Synthesis of small    RNAs using T7 RNA polymerase. Methods in Enzymology, 180, 51-62.-   22. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. and    Stuber, D. (1988) Genetic approach to facilitate purification of    recombinant proteins with a novel metal chelate adsorbent.    Bio-Technol, 6, 1321-1325.-   23. Cleary, M. A., Kilian, K., Wang, Y. Q., Bradshaw, J., Cavet, G.,    Ge, W., Kulkarni, A., Paddison, P. J., Chang, K., Sheth, N. et    al. (2004) Production of complex nucleic acid libraries using highly    parallel in situ oligonucleotide synthesis. Nat. Methods, 1,    241-248.-   24. Gao, X., Gaffney, B. L., Senior, M., Riddle, R. R. and    Jones, R. A. (1985) Methylation of thymine residues during    oligonucleotide synthesis. Nucleic Acids Res, 13, 573-584.-   25. Pon, R. T., Damha, M. J. and Ogilvie, K. K. (1985) Modification    of guanine bases by nucleoside phosphoramidite reagents during the    solid phase synthesis of oligonucleotides. Nucleic Acids Res, 13,    6447-6465.-   26. Pon, R. T., Usman, N., Damha, M. J. and Ogilvie, K. K. (1986)    PREVENTION OF GUANINE MODIFICATION AND CHAIN CLEAVAGE DURING THE    SOLID-PHASE SYNTHESIS OF OLIGONUCLEOTIDES USING PHOSPHORAMIDITE    DERIVATIVES. Nucleic Acids Res, 14, 6453-6470.-   27. Crippa, S., Digennaro, P., Lucini, R., Orlandi, M. and    Rindone, B. (1993) CHARACTERIZATION OF ADDUCTS OF NUCLEIC BASES AND    ACRYLIC-MONOMERS. Gazzetta Chimica Italiana, 123, 197-203.-   28. Prime, K. L. and Whitesides, G. M. (1993) Adsorption of Proteins    onto Surfaces Containing End-Attached Oligo(Ethylene Oxide)—a Model    System Using Self-Assembled Monolayers. Journal of the American    Chemical Society, 115, 10714-10721.-   29. Pease, A. C., Solas, D., Sullivan, E. J., Cronin, M. T.,    Holmes, C. P. and Fodor, S.P.A. (1994) Light-Generated    Oligonucleotide Arrays for Rapid DNA-Sequence Analysis. P Natl Acad    Sci USA, 91, 5022-5026.-   30. McGall, G. H., Barone, A. D., Diggelmann, M., Fodor, S. P. A.,    Gentalen, E. and Ngo, N. (1997) The efficiency of light-directed    synthesis of DNA arrays on glass substrates. J Am Chem Soc, 119,    5081-5090.-   31. Walbert, S., Pfleiderer, W. and Steiner, U. E. (2001)    Photolabile protecting groups for nucleosides: Mechanistic studies    of the 2-(2-nitrophenyl)ethyl group. Helvetica Chimica Acta, 84,    1601-1611.

1. An RNA-mediated assembly method for providing a target RNA molecule,comprising: (a) providing a: (i) a plurality of first oligonucleotideseach having a segment sequence corresponding to a portion of a targetRNA and a complementary RNAP promoter sequence operably-linked to saidsegment sequence's 3′ termini; and (ii) a plurality of secondoligonucleotides each having a splint sequence corresponding to aportion of the target RNA that complements and partially overlaps thesegment sequence of the first surface-bound oligonucleotides, saidsecond surface-bound oligonucleotides including an RNA polymerasepromoter sequence operably-linked to their splint sequence's 3′ termini;(b) hybridizing a third oligonucleotide comprising a sequencecomplementary to the RNAP promoter sequence of the first and secondpluralities of oligonucleotides to yield double-stranded RNAP promoters;(c) transcribing with RNA polymerase, in the presence of rNTPs, thesegment sequences of the plurality of first oligonucleotides to yield anRNA segment and the splint sequences of the plurality of secondoligonucleotides to yield an RNA splint; (d) assembly of the RNAsegments and RNA splints by hybridization to form RNA:RNA hybrids; and(e) sealing nicks in the RNA:RNA hybrid to provide a target RNAmolecule.
 2. The method according to claim 1, wherein the plurality offirst oligonucleotides or the plurality of second oligonucleotides isprovided as a surface-bound oligonucleotide array.
 3. The methodaccording to claim 1, wherein in step (c), transcription is carried outin the presence of a mixture of rNTPs and rNMPs.
 4. The method accordingto claim 1, further comprising removing terminal pyrophosphate groupsfrom the RNA segments prior to step (e).
 5. The method according toclaim 4, wherein removal of the terminal pyrophosphate groups isperformed by treatment with 5′ pyrophosphohydrolase or RNApyrophosphatase.
 6. The method according to claim 1, wherein the splintsequences are transcribed in the presence of 2′-O-Methyl ribonucleotidesand sealing the nicks in step (e) is carried out with T4 DNA ligase or atruncated version thereof.
 7. The method according to claim 1, whereinthe target RNA molecule is a full-length RNA transcript of a gene. 8.The method according to claim 2, wherein the oligonucleotide array'ssurface comprises a silanized glass or amorphous carbon deposited on agold film.
 9. The method according to claim 2, wherein theoligonucleotide array is provided by in situ photolithographicoligonucleotide array synthesis.
 10. The method according to claim 2,wherein said surface-bound oligonucleotides include a spacer, a T7 RNAPpromoter sequence, a CC dinucleotide and either the segment sequence orthe splint sequence.
 11. The method according to claim 1, wherein thethird oligonucleotide includes a T7 RNAP promoter sequence and adinucleotide GG.
 12. The method according to claim 1, wherein step (c)results in the synthesis of multiple copies of RNA segments and RNAsplints from each of the segment sequences and splint sequences.